The use of microfabricated and microfluidic structures in biomedical research has been rapidly expanding in recent years, but most research applications of these devices require customization, and new applications typically require several design iterations for troubleshooting. In an effort to bring the necessary technical capabilities and knowledge to do so into biomedical laboratories, we have developed a basic in-house microfabrication facility accessible to researchers across the intramural program. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. We are able to reliably pattern single- and multi-layer template features with lateral dimensions of less than 2 microns, and with heights ranging from a few microns to a few hundred microns. We have developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels, including techniques for making and manipulating thin (<200 micrometer) PDMS layers for use in multilayer devices and bottomless structures. We can also perform surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass, and have developed techniques for connecting devices to flow-control instruments such as syringe pumps and pressure controllers. We also have protocols for generating micropatterns of biomolecules on surfaces using microcontact printing or selective UV exposure, as well as protocols for generating monodisperse droplets and hydrogel beads. In addition, we have the ability to deposit and pattern metal layers, as well as a heated hydraulic press for hot-embossing thermoplastics, including PMMA, polycarbonate, and COC. We have also used a programmable razor cutter and pressure-sensitive adhesive to directly make thin film structures with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This is a low-cost and convenient method for several applications, including the ready fabrication of flow cells with two glass walls, or for fluidic confinement over already-functionalized surfaces. Finally, we continue to work on finite element modeling of transport in microfabricated structures, developing models for oxygen delivery in a bioreactor with micropillars and for chemokine concentration in a microfluidic hydrogel device. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NEI, NIAID, NHLBI, NCI, NICHD, NINDS, NIDCD, NIDDK, and NIBIB. 1) A collaborative effort with LSB, NIAID, to study chemotaxis, currently focusing on the decision-making of single cells exposed to competing solution gradients. Earlier efforts with this group looked at chemotaxis of primary immune cells in 3-D collagen matrices, using a microfluidic agarose device to generate reproducible time-varying spatial gradients on a platform compatible with high-resolution fluorescence and two-photon imaging. 2) The design, fabrication, modeling, and use of an oxygen-transmissive membrane, patterned with a micropillar array to deliver oxygen to three-dimensional cell culture volumes with in vivo-like spatial distribution, in collaboration with LCB, CCR, NCI and SPIS, CIT. Because the pillar spacing is approximately equal to typical intercapillary distances (200 microns), cells in a Matrigel layer surrounding the pillars can be maintained under hypoxic conditions in an extended 3D volume. We have recently started working with researchers in LAMB, NHLBI to employ their recently developed FLIM-FRET probe for intracellular oxygen to directly image spatial and cellular variations in oxygenation in these structures. 3) The fabrication and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, light-activated microdissection. This work was begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD; more recent efforts with NCI and CIT have focused on refining the instrumentation and developing standardized protocols for a variety of targets and stain localizations. 4) The use of a microfluidic channel together with custom instrumentation developed in LCMB, NICHD in order to separate the effects of pressure and rapid (msec) shear transients on cultured neural cells, in order to gain a better understanding of the cellular mechanisms of traumatic brain injury. Our group has fabricated the microchannels, performed modelling aimed at understanding the shear transients experienced by the cells as a function of the bulk flow, and consulted extensively on how best to interface the microchannel with the existing instrumentation. 5) At the instigation of LCIMB, NIBIB, the collaborative design and fabrication of an aluminum-patterned window for use in calibrating the measured settling distances in analytical ultracentrifuges. NIST researchers are currently working on the development and validation of a low-volume, commercially available, certified standard, based on the prototype windows made here. 6) In collaboration with LCB, NCI, development of an in vitro model to complement measurements on cell migration in zebrafish to advance understanding of the mechanisms for cell migration and metastasis. 7) In collaboration with researchers in NINDS and NICHD, the development of devices to enable selective harvesting of axons from cultured neurons for biochemical analysis, in order to readily be able to generate enough material for downstream measurements. 8) In collaboration with researchers from the LIG, NIAID, the development of PDMS/glass microfluidic devices for studying chemotaxis. The first device design enables measurements of chemotaxis in 2D chambers on a platform compatible with high-resolution imaging; the generated gradients can be directly measured with a fluorescent tracer, and the system operates without pumps.